The present invention is concerned with a carrier-bound, multicomponent detection system for the colorimetric determination of esterolytically- and/or proteolytically-active ingredients of body fluids.
The detection of esterolytically- and/or proteolytically-active ingredients of body fluids is of great importance, especially in diagnosis of diseases of the kidneys and of the urogenital tract. In these cases, it is important to detect leukocytes in urine on the basis of their inherent esterolytic and/or proteolytic activity. Carrier-bound detections systems, for example in the form of test strips, have proven to be especially advantageous for this purpose because these only need to be dipped briefly into the liquid sample to be investigated and then subsequently, after removal of the strip from the sample, permit a determination of the number of leukocytes/ml of sample, for example on the basis of color formed, within a short period of time. It is thus possible to provide a clean, simple and rapid method for the detection of such ingredient materials in body fluids without complicated time- and cost-consuming manipulations having to be carried out with or on the sample for this purpose.
From the prior art, so-called multicomponent systems are taught as being advantageous detection systems for the colorimetric determination of esterolytically- and/or proteolytically-active ingredients of body fluids, for example of leukocytes, in urine. These multicomponent systems are those reagent systems which comprise several chemical substances, at least one of which is so changed by esterolytically- and/or proteolytically-active ingredients materials of body fluids that the reaction product reacts directly with another component of the detection system or reacts in a reaction sequence with several of the other components of the detection system to give a colored substance which can then be determined colorimetrically. The amount of colored substance formed is a measure of the amount of esterolytically- and/or proteolytically-active ingredients in the sample investigated.
Especially suitable for this type of detection are two-component systems in which an ester is first cleaved by the ingredient materials to be detected, followed by reaction of the resultant hydroxy compound with a further reactive substance to give a colored material. Carrier-bound multicomponent systems based upon this principle are known, for example, from U.S. Pat. Nos. 4,551,428 and 4,749,648.
These patents concern, inter alia, a carrier-bound detection system in which an ester and a diazonium salt are present on the same reagent layer. The ester reacts with a ingredient of body fluid, such as urine, which is esterolytically and/or proteolytically active. The product of the reaction is a hydroxy compound, which then reacts with the diazonium salt, forming a colored product. The joint presence of all components of the detection system on one and the same reagent layer appears to be optimal insofar as it appears to be ensured that the product resulting by reaction with the esterolytically- and/or proteolytically-active component can quickly react further to give a colored end product.
Rapid and complete detection reactions form the basis for a sensitive test. In the case of the above-mentioned detection system, the detection limit for leukocytes in urine for reading off the value after about 2 minutes is about 15 to 25 leukocytes/ul of urine. However, from the point of view of the physician, it is desirable to have available detection agents which are even more sensitive. Comparatively long waiting periods for reading off the value to be determined are undesirable.